Protein-protein interaction between Fli-1 and GATA-1 mediates synergistic expression of megakaryocyte-specific genes through cooperative DNA binding
Protein-protein interaction between Fli-1 and GATA-1 mediates synergistic expression of megakaryocyte-specific genes through cooperative DNA binding
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DOI：
10.1128/mcb.23.10.3427-3441.2003
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发表时间：
2003-05-01
影响因子：
5.3
通讯作者：
Chong, BH
中科院分区：
文献类型：
--
作者：
Eisbacher, M;Holmes, ML;Chong, BH
Friend白血病整合蛋白1(Fli-1)是ETS转录激活子家族的成员之一，已被证明是巨核细胞分化过程中的重要调节因子。我们进行了K562基因文库的双杂交筛选，以确定与Fli-1相互作用的转录因子，这些转录因子可能是巨核细胞发育的调节因子。在这里，我们报道了Fli-1与GATA-1的物理相互作用，GATA-1是一种功能齐全的锌指转录因子，对红系和巨核细胞的分化都至关重要。我们绘制了相互作用所需的最小结构域，并表明GATA-1的锌指与Fli-1的Ets结构域相互作用。GATA-1先前已被证明与Fli-1相关蛋白PU.1的Ets结构域相互作用，这两种蛋白似乎相互抑制对方的活性。相反，我们证明了GATA-1和Fli-1在瞬时转染中协同激活巨核细胞特异性启动子Gpix和GPIbalpha。用含有ETS和GATA结合基序的Gpix启动子衍生的寡核苷酸的定量凝胶迁移率分析表明，Fli-1和GATA-1表现出协同的DNA结合，其中GATA-1与DNA的结合在Fli-1的存在下增加了约26倍(从4.2 nM增加到0.16 nM)，这为观察到的转录协同作用提供了一种机制。为了测试对内源性基因的影响，我们在富含GATA-1的K562细胞中稳定过表达Fli-1。Northern印迹和荧光激活细胞分类分析表明，Fli-1过表达诱导内源性Gpix和GPIbalpha基因的表达。这项工作表明，Fli-1和GATA-1共同作用于激活巨核细胞终末分化相关基因的表达。
Friend leukemia integration 1 (Fli-1) is a member of the Ets family of transcriptional activators that has been shown to be an important regulator during megakaryocytic differentiation. We undertook a two-hybrid screen of a K562 cDNA library to identify transcription factors that interacted with Fli-1 and were potential regulators of megakaryocyte development. Here we report the physical interaction of Fli-1 with GATA-1, a well-characterized, zinc finger transcription factor critical for both erythroid and megakaryocytic differentiation. We map the minimal domains required for the interaction and show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. GATA-1 has previously been shown to interact with the Ets domain of the Fli-1-related protein PU.1, and the two proteins appear to inhibit each other's activity. In contrast, we demonstrate that GATA-1 and Fli-1 synergistically activate the megakaryocyte-specific promoters GPIX and GPIbalpha in transient transfections. Quantitative electrophoretic mobility shift assays using oligonucleotides derived from the GPIX promoter containing Ets and GATA binding motifs reveal that Fli-1 and GATA-1 exhibit cooperative DNA binding in which the binding of GATA-1 to DNA is increased approximately 26-fold in the presence of Fli-1 (from 4.2 to 0.16 nM), providing a mechanism for the observed transcriptional synergy. To test the effect on endogenous genes, we stably overexpressed Fli-1 in K562 cells, a line rich in GATA-1. Overexpression of Fli-1 induced the expression of the endogenous GPIX and GPIbalpha genes as measured by Northern blot and fluorescence-activated cell sorter analysis. This work suggests that Fli-1 and GATA-1 work together to activate the expression of genes associated with the terminal differentiation of megakaryocytes.